This project's overall aim is to understand the structure of eukaryotic tRNA genes, both in relation to the functional constraints on these genes and to the evolutionary history of the multi-gene families to which they belong. The experiments will involve yeast nonsense suppressor tRNATyr and tRNASer genes, all of which are already well defined both genetically and molecularly. Experiments will focus on two areas - the role of 5 feet-flanking sequences in tRNA gene expression and the mechanism of the sequence exchanges that are presumed to occur at a low frequency between different members of a dispersed tRNA gene family. The emphasis on 5 feet-flanking sequences is motivated by two observations: 1) Highly specific naturally occurring polymorphisms are commonplace in these regions; 2) The construction of in vitro deletion-fusions upstream from the coding region can severely impair in vivo gene function. The project will determine whether or not the naturally occurring polymorphisms, particularly those associated with the sigma insertion sequence, affect gene expression by examining their effects on the phenotype of an adjacent suppressor gene. The molecular basis of the expression defect caused by the deletion-fusions will be explored by a combination of in vitro transcription experiments and genetic reversion analysis. Finally, the factors influencing inter-locus sequence exchanges will be examined by observing the movement of sequences within a gene family that contains a single functional or non-functional tyrosine-inserting UGA suppressor. This system allows selections for extremely rare genetic events since these suppressors cannot arise from wild type tRNATyr genes by a single mutation. We also plan to exploit our experience with nonsense suppressor genes in transformation vectors to develop new genetic methods that will extend the overall utility of nonsense mutations in yeast genetics.